Neisseria gonorrhoeae (Ng) is the causative agent of the sexually transmitted infection, gonorrhea. Gonorrhea causes over 100 million new cases worldwide annually and adversely affects the reproductive health of women. Ng has become resistant to almost every antibiotic that has been used and has now achieved superbug status. Novel vaccines and therapies against this infection are urgently needed. Complement (C) is a critical arm of innate immune defenses against Ng. Ng possesses several mechanisms to subvert C activation and function, including binding of C inhibitors such as factor H (fH) and C4b-binding protein (C4BP) and blocking antibody (Ab) directed against a ubiquitous conserved outer membrane protein called reduction modifiable protein (Rmp). Gonorrhea is characterized by an influx of polymorphonuclear leukocytes (PMNs) into the genital tract, which in concert with Ab and C may facilitate clearance of infection. In Specific Aim 1a, we will elucidate the roles of PMNs, C and C receptors in vaccine efficacy. Our vaccine candidate is a peptide mimic (mimitope) of a Ng lipooligosaccharide epitope that elicits bactericidal and opsonophagocytic Ab in mice. Because binding of fH and C4BP to gonococci is human-specific, experiments will be carried out in human fH+/C4BP+ Tg mice, which we believe will more faithfully simulate protection in humans. Vaccine Ab efficacy will be evaluated in fH+/C4BP+ Tg mice that lack either PMNs, C3 (opsonin) or C5a (chemotaxin) receptor. In Specific Aim 1b, we will compare temporal changes in the vaginal transcriptome of immunized mice with unimmunized control mice (both infected with Ng) to define transcriptional profiles associated with vaccine- mediated bacterial clearance. Vaccine development against Ng has been hindered by lack of knowledge of the correlates of protection and these data using an unbiased genome-wide approach will inform rational vaccine development. In Specific Aim 2, we will test the efficacy of a novel immunotherapeutic that binds to and kills Ng by Fc-mediated C activation. We developed the therapeutic based our studies that showed binding of the 3 C-terminal domains of fH to Ng. These domains were fused to IgG Fc to create the therapeutic molecule. Because the C-terminus of native fH binds to host glycosminoglycans (and limits toxic C activation on host cells), we created a mutation in the fH portion of the fH/Fc molecule that abrogates binding to the host, but not to the gonococcal surface thereby maintaining full C-mediated killing of Ng. We will test our lead fH/Fc molecule against antibiotic-resistant Ng in a murine vaginal infection model using fH+/C4BP+ Tg mice. Mice will also be evaluated for renal, ocular and hematologic toxicity. In Specific Aim 3 we will determine how Abs directed against Ng Rmp that blocks C-dependent Ab killing of gonococci and negatively impact the efficacy of vaccine Ab subverts complement-mediated bactericidal function by vaccine Ab. The molecular basis of blocking of C-mediated killing of Ng will be elucidated. Completion of the proposed studies will represent an important step forward in the development of novel vaccines and therapies against gonorrhea.